Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K⁺ and Na⁺ affinity of Na,K-ATPase (Crambert et al., 2002, Proc. Natl. Acad. Sci. USA 99, 11476-11481). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinase A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity, or on the apparent K+ affinity of Na,K-ATPase 1/1 and 2/1 isozymes but increases their apparent Na⁺ affinity, dependent on PLM phosphorylation at Ser68. Phosphorylation of phospholemman by protein kinase C does neither affect the maximal transport activity of 1/1 isozymes nor the K⁺ affinity of 1/1 and 2/1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of 2/1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase 1/ and 2/ isozymes and increases their apparent Na⁺ affinity whereas protein kinase C phosphorylation of PLM modulates the transport activity of Na,K-ATPase 2/ but not of 1/ isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.